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Biomedical subjects

G M Rubanyi

Publications and source records attributed to G M Rubanyi.

At least 37 records · Page 2Linked to original sources
PubMed1997

Nitroxides increase the detectable amount of nitric oxide released from endothelial cells.

Nitroxides are known to exert superoxide dismutase-mimetic properties and to decrease O-2- and H2O2-mediated cytotoxicity. However, the effect of nitroxides on .NO homeostasis has not been studied yet. The present study investigates the effect of nitroxides on the detectable amount of .NO released by 3-morpholinosydnonimine (SIN-1) and cultured endothelial cells. Cultured bovine aortic and atrial endothelial cells stimulated with 10 microM A23187 released a stable flux of .NO, as detected by .NO chemiluminescence. Addition of 100 units/ml SOD or 10 microM of the nitroxides 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPOL), 3-carboxy-proxyl, and 3-ethoxycarbonyl-proxyl, increased the chemiluminescence signal. The effect of these nitroxides on the amount of .NO released from cell monolayers was dose-dependent, with the highest efficacy between 30 and 100 microM. EPR spin trapping in SIN-1 solutions revealed the formation of .OH adducts from spontaneous dismutation of O-2 and concomitant reaction with H2O2. Both SOD and TEMPOL increased the signal intensity of the .OH adduct by accelerating the dismutation of O-2. The results of this study demonstrate that the SOD-mimetic activity of nitroxides increases the amount of bioavailable .NO in vitro.

Animals↗
PubMed1997

Vascular estrogen receptors and endothelium-derived nitric oxide production in the mouse aorta. Gender difference and effect of estrogen receptor gene disruption.

The present study was designed to test the hypothesis that estrogen receptors (ER) in the blood vessel wall play a role in the modulation of the release of endothelium-derived nitric oxide (EDNO). Both basal and stimulated release of EDNO were determined in aortic rings isolated from female and male wild-type and male homozygous estrogen receptor knock-out (ERKO) mice. 125I-17beta-estradiol binding in aortic tissue showed significantly more high affinity cytosolic- nuclear-binding sites in male compared with female wildtype mice. Estrogen receptor transcripts were present in the aorta of male wild-type mice, but they were absent in male ERKO animals. Basal release of EDNO (determined by endothelium-dependent contraction caused by NG-nitro-arginine) was significantly higher in aorta of wild-type male mice compared with wild-type female mice, and significantly lower in the aorta of male ERKO compared with male wild-type mice. Acetylcholine-induced endothelium-dependent relaxation was similar in all groups studied. No difference was observed in the activity of calcium-dependent nitric oxide synthase in homogenates of lungs and brain taken from male wild-type and ERKO mice. These studies show a significant association between the number of estrogen receptors and basal release of EDNO in the aorta of mice, and suggest that decreased vascular estrogen receptor number may represent a novel risk factor for cardiovascular diseases.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1997

Quantitative measurement for endothelial constitutive nitric oxide synthase in cultured human endothelial cells.

Constitutively expressed endothelial nitric oxide synthase (ecNOS) produces nitric oxide (NO) from L-arginine and is important for the maintenance of cardiovascular homeostasis. We report the development of a capture ELISA which is specific for ecNOS. The assay detection limit is 0.5 ng/ml ecNOS protein, allowing the measurement of ecNOS from as few as 6000 human endothelial cells cultured in 96-well microtiter plates. This ELISA has been used to measure a downregulation of ecNOS with 24-h TNFalpha treatment, consistent with results obtained by Western blot analysis. Quantitation of ecNOS in human endothelial cells showed a higher expression of ecNOS in human aortic endothelial cells (18.3+/-1.35 ng ecNOS per 10(6) cells, n = 3 donors) than in human umbilical vein endothelial cells (10.4+/-0.48 ng ecNOS per 10(6) cells, n = 3 donors). These studies demonstrate that this convenient, quantitative assay is currently the most sensitive method for investigating ecNOS protein regulation.

Amino Acid Sequence↗
PubMed1997

Increased expression of the cardiac L-type calcium channel in estrogen receptor-deficient mice.

Steroid hormones control the expression of many cellular regulators, and a role for estrogen in cardiovascular function and disease has been well documented. To address whether the activity of the L-type Ca2+ channel, a critical element in cardiac excitability and contractility, is altered by estrogen and its nuclear receptor, we examined cardiac myocytes from male mice in which the estrogen receptor gene had been disrupted (ERKO mice). Binding of dihydropyridine Ca2+ channel antagonist isradipine (PN200-110) was increased 45.6% in cardiac membranes from the ERKO mice compared to controls, suggesting that a lack of estrogen receptors in the heart increased the number of Ca2+ channels. Whole-cell patch clamp of acutely dissociated adult cardiac ventricular myocytes indicated that Ca2+ channel current was increased by 49% and action potential duration was increased by 75%. Examination of electrocardiogram parameters in ERKO mice showed a 70% increase in the QT interval without significant changes in PQ or QRS intervals. These results show that the membrane density of the cardiac L-type Ca2+ channel is regulated by the estrogen receptor and suggest that decreased estrogen may lead to an increase in the number of cardiac L-type Ca2+ channels, abnormalities in cardiac excitability, and increased risk of arrhythmia and cardiovascular disease.

Action Potentials↗
PubMed1997

17 beta-Estradiol attenuates endotoxin-induced excessive nitric oxide production in ovariectomized rats in vivo.

Experiments were designed to examine the effect of 17 beta-estradiol on lipopolysaccharide (LPS)-induced excessive nitric oxide production in vivo. Ovariectomized and sham-operated female rats were injected with LPS (5 mg/kg ip), and different groups of ovariectomized, LPS-injected animals were treated with either 17 beta-estradiol, dexamethasone, or aminoguanidine. Nitric oxide generation was estimated by measuring plasma nitrite levels 12 h after LPS injection. LPS treatment of the rats resulted in a four- to fivefold increase in circulating plasma nitrite level, significantly higher in the ovariectomized animals compared with the sham-operated animals. The LPS-induced plasma nitrite increase could be prevented by dexamethasone (3 mg/kg ip) injected 1 h before LPS treatment. 17 beta-Estradiol (3 micrograms/rat sc), administered 48 h before LPS treatment, significantly attenuated the LPS-induced elevation in plasma nitrite levels. This effect was comparable to that achieved by aminoguanidine (200 microM/kg), an inhibitor of inducible nitric oxide synthase, injected 1 h after LPS treatment. The present findings suggest that estrogens may be beneficial in pathological conditions associated with excessive nitric oxide generation.

Analysis of Variance↗
PubMed1997

Effects of estrus cycle, ovariectomy, and treatment with estrogen, tamoxifen, and progesterone on apolipoprotein(a) gene expression in transgenic mice.

Plasma levels of lipoprotein(a) (Lp(a)), are regulated by the synthetic rate of apolipoprotein(a) (apo(a)), a major protein component of this atherogenic lipoprotein. Exogenously administered sex steroid hormones are potent regulators of plasma Lp(a) concentrations. We utilized a recently developed apo(a) yeast artificial chromosome (YAC) transgenic mouse model to study the effects of ovariectomy, estrus cycle, and exogenous administration of ethinyl-estradiol, the partial estrogen receptor agonist, tamoxifen, and progesterone on circulating apo(a) plasma levels. Analysis of liver RNA revealed that estrogen and tamoxifen exerts their plasma apo(a) lowering effect at the level of apo(a) mRNA. This action of estrogen and tamoxifen may contribute to their antiatherosclerotic and cardiovascular protective effect.

Animals↗
PubMed1997

Mechanism of vascular smooth muscle relaxation by estrogen in depolarized rat and mouse aorta. Role of nuclear estrogen receptor and Ca2+ uptake.

17 beta-Estradiol induces vasodilation in vitro and in vivo, which has been suggested to contribute to the cardiovascular protection by this ovarian steroid hormone. However, the exact mechanism of vasorelaxation by estrogens remains to be elucidated. In this study, we analyzed the potential role of genomic mechanisms involving the nuclear estrogen receptor and inhibition of entry of extracellular Ca2+ in 17 beta-estradiol-induced vasorelaxation in depolarized aortic rings, isolated from male and female rats and male mice. In both male and female rat aortic rings without endothelium and in intact male mouse aortic rings treated with NG-nitro-L-arginine, 17 beta-estradiol caused dose-dependent (0.3 to 30 mumol/L) relaxation of contraction evoked by high-K+ depolarization (30 and 45 mmol/L KCl, respectively). The estrogen receptor antagonist ICI 164384 had no effect on 17 beta-estradiol-induced relaxations. 125I-17 beta-estradiol binding studies showed the presence of high-affinity cytosolic-nuclear estrogen receptors in control male mouse aortas. Comparable relaxations of aortic rings isolated from control and estrogen receptor-deficient transgenic mice provided direct evidence that the nuclear estrogen receptor is not involved in this response. 17 beta-Estradiol-induced relaxation of rat aortic rings could not be prevented by cycloheximide or actinomycin D, suggesting that the response was not mediated by de novo protein synthesis or gene transcription. In rat aortic rings, 17 beta-estradiol inhibited the increase of 45Ca uptake by 30 mmol/L KCl at concentrations (10 and 30 mumol/L) that caused vasorelaxation in the same tissue, suggesting that inhibition of Ca2+ entry contributes to the response. 17 alpha-Estradiol was less effective, and estrone was devoid of vasorelaxing activity. Vasorelaxation by estrogens in female and male rat aortas was similar, indicating no gender difference in vascular responses under these conditions.

Action Potentials↗
PubMed1996

Disruption of estrogen receptor gene prevents 17 beta estradiol-induced angiogenesis in transgenic mice.

Estrogen is known to modulate angiogenesis, both under physiological and pathological conditions, and has been demonstrated to augment angiogenesis induced by bFGF in a mouse model. We have modified this mouse model and measured the apparent plasma volume in Matrigel plugs containing basic fibroblast growth factor (bFGF) in wild type and estrogen receptor knockout, ovariectomized mice in the presence and absence of exogenous 17 beta estradiol. The apparent plasma volume was determined by measuring the fluorescence of the excised plug 10 min. after injection of fluoroscein labeled dextran 150. In wild type mice exogenous 17 beta estradiol increased the apparent plasma volume of the Matrigel plug and the uterine weight significantly. In the estrogen receptor knockout mice exogenous 17 beta estradiol caused a small, but significant increase in uterine weight but was without effect on the apparent plasma volume of the Matrigel plug. It is concluded that functional estrogen receptors are essential for the augmentation of bFGF-induced angiogenesis by exogenous 17 beta estradiol in female mice.

Animals↗
PubMed1995

Gender difference in endothelial dysfunction in the aorta of spontaneously hypertensive rats.

We investigated endothelium-dependent responses of thoracic aorta isolated from age-matched male and female spontaneously hypertensive rats (SHR) to explore gender differences in endothelial dysfunction that may contribute to the sexual dimorphism observed in the development of hypertension in this strain. Endothelium-dependent relaxation in response to acetylcholine (10(-9) to 10(-4) mol/L) was significantly greater in female rats than in male rats, although impaired responses were seen in both sexes compared with normotensive controls. Inhibition of cyclooxygenase by indomethacin (10(-5) mol/L) improved endothelium-dependent relaxation, but it did not abolish the gender difference. Relaxations in response to sodium nitroprusside were identical in denuded aortic rings from male and female SHR. Acetylcholine at higher concentrations (10(-6) to 10(-4) mol/L) induced endothelium-dependent contraction in intact, quiescent aortic rings from male SHR but not in those from female SHR. After incubation with NG-nitro-L-arginine (10(-4) mol/L), contraction in response to acetylcholine became apparent in rings from female SHR, but it was still significantly less pronounced than in similarly treated rings from male SHR. Endothelium-dependent contraction was prevented by indomethacin in both sexes, suggesting that a cyclooxygenase product such as endoperoxide may be mediating this effect. Because responses evoked by the thromboxane/endoperoxide receptor agonist U46619 (10(-10) to 10(-6) mol/L) were not greater in rings from male SHR than those from female SHR, increased smooth muscle responsiveness or higher thromboxane/endoperoxide receptor density in the males could not account for the differences in endothelium-dependent contraction. These results suggest that sex steroid hormones may control endothelium-dependent vascular reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1994

Effects of NW-nitro-L-arginine and dexamethasone on early events following lipopolysaccharide injection: observations in the hamster cheek pouch microcirculation.

The effects of NW-nitro-L-arginine (L-NAG) and dexamethasone in the microcirculatory changes observed in early stages of endotoxemia was investigated in male hamsters treated with Escherichia coli lipopolysaccharide (LPS). The cheek pouch was studied in vivo by means of intravital microscopy and mean arterial and venous pressures, mean arteriolar internal diameter, spontaneous arteriolar vasomotion, microvascular blood flow, macromolecular permeability, leukocyte adhesion, and mean survival time were evaluated in animals treated with either LPS alone or the combination of LPS with L-NAG, an inhibitor of both the constitutive and inducible NO synthases (NOs). The intravenous injection of LPS (100 mg/kg) elicited a significant reduction in mean arterial blood pressure (MABP) and arteriolar blood flow. The observed arterioles dilated and the spontaneous vasomotion ceased. The combination LPS + L-NAG, both given intravenously, prevented the reduction of MABP and the vasodilation but did not help either the reduction of arteriolar blood flow or the cessation of vasomotion. In order to separate the effect of the two NOs, a group of hamsters was pretreated with dexamethasone (10 mg/kg, also intravenously) which inhibits the induction of the inducible NO synthase (iNOs). In this group, the hypotension, vasodilation, and cessation of vasomotion were prevented but the decrease in arteriolar blood flow was not affected. The mean survival time was significantly decreased by the combination of LPS + L-NAG (35 +/- 6 h) and significantly increased by the pretreatment with dexamethasone (92 +/- 5 h) compared to LPS alone (56 +/- 7 h).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Gender difference in bioassayable endothelium-derived nitric oxide from isolated rat aortae.

Gender differences in the production/release of endothelium-derived nitric oxide (EDNO) was assessed by determining the ability of intact endothelium to suppress serotonin- (10(-7)-10(-5) M) and phenylephrine-induced (10(-9)-(10(-5) M) contractions in thoracic aortae isolated from male and female Wistar rats mounted in organ chambers for isometric tension recording or tested in bioassay experiments. The endothelium suppressed these contractions significantly more in aortae from female than from male rats. In the bioassay, the perfusate from intact female thoracic aortic segments produced a significantly greater relaxation of the detector rings than that from the aortae isolated from male rats. Acetylcholine (10(-9)-10(-5) M), used to investigate agonist-induced release of EDNO, evoked significantly greater endothelium-dependent relaxation in aortae from female rats. The unstimulated release of 6-ketoprostaglandin F1 alpha and thromboxane B2 from intact thoracic aortic rings from male and female rats was not significantly different. There was no difference in smooth muscle reactivity to sodium nitroprusside (10(-10)-10(-6) M) in rings without endothelium. These results indicate that EDNO production/release is higher in thoracic aortae isolated from female rats.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

The role of endothelium in cardiovascular homeostasis and diseases.

Key discoveries in the past decade have revealed that the vascular endothelium is an important regulatory organ that is involved in maintaining cardiovascular homeostasis in health and contributes significantly to the pathomechanism of several cardiovascular diseases. Occupying a strategically important location between circulating blood and tissues and having the ability to respond to changes in its physical, chemical, and humoral environment by the production of a host of biologically active substances, the normal endothelium modulates the tone of underlying vascular smooth muscle, maintains a nonadhesive luminal surface, and mediates hemostasis, cellular proliferation, and inflammatory and immune mechanisms in the vascular wall. Modulation of smooth-muscle tone is mediated by the synthesis release of endothelium-derived relaxing [PGI2, EDRF(NO), and EDHF] and contracting factors (arachidonic acid metabolites, endothelin-1, and angiotensin II). Anticoagulant, fibrinolytic, and antithrombotic properties contribute to the maintenance of the fluidity of blood. Injury or activation (by cytokines) of endothelial cells disrupts these normal regulatory mechanisms and results in morphologic and functional alterations (phenotypic changes) commonly defined as endothelial dysfunction. Clinically, the "syndrome" of endothelial cell dysfunction can be described as generalized or localized vasospasm, thrombosis, atherosclerosis, and restenosis. Although its importance is clearly established, no drugs used today were originally targeted for the treatment of endothelial dysfunction. Recent studies, however, showed that some existing therapies (e.g., angiotensin-converting enzyme inhibitors) may protect the endothelium. Novel diagnostic techniques and innovative therapeutic strategies, based on the already known molecular mechanisms of endothelial dysfunction, are briefly outlined. Further knowledge of the pathobiology of the impaired endothelium will contribute to unraveling some of the remaining mysteries of many cardiovascular diseases and will enable us to design novel therapies to prevent and treat them.

Animals↗
PubMed1993

Effect of cilazapril and indomethacin on endothelial dysfunction in the aortas of spontaneously hypertensive rats.

Chronic hypertension is associated with impaired endothelial function [i.e., reduced synthesis/release of endothelium-derived relaxing factor (EDRF) and increased synthesis/release of endothelium-derived contracting factor (EDCF)] in both animals and humans. Although it is not known whether endothelial dysfunction is the consequence and/or an important pathogenetic cause of hypertension, the goal of effective antihypertensive therapy should include restoration of normal endothelial function as well. Because angiotensin I-converting enzyme (ACE) inhibitors are effectively used in the treatment of hypertension, the aim of the present study was to test whether in vivo treatment of spontaneously hypertensive rats (SHRs) with the ACE inhibitor cilazapril improves endothelial function in the isolated thoracic aorta of SHRs. Treatment of SHRs with cilazapril (10 mg/kg/day orally for 2 weeks) resulted in a significant decrease in blood pressure and normalization of endothelium-dependent relaxation evoked by acetylcholine (ACh) and adenosine diphosphate (ADP). However, cilazapril treatment had no significant effect on endothelium-dependent contractions evoked by 5-hydroxytryptamine (5-HT; serotonin) and prostaglandin F2 alpha (PGF2 alpha). In contrast, in vitro treatment of isolated thoracic aortas with indomethacin (10(-5) M) normalized endothelium-dependent relaxations to ACh and ADP as well as inhibited endothelium-dependent contractions to 5-HT and PGF2 alpha. These results suggest that the ACE inhibitor cilazapril increases the synthesis/release of EDRFs whereas indomethacin prevents the synthesis/release of cyclo-oxygenase-derived EDCFs in the endothelium of rat aorta. The exact mechanism of action of ACE inhibitors on endothelial dysfunction remains to be determined.

Acetylcholine↗
PubMed1992

Effects of intracoronary administration of endothelin in anesthetized dogs: comparison with Bay k 8644 and U 46619.

The effects of synthetic endothelin on the coronary circulation were studied in pentobarbital-anesthetized dogs and compared with those of Bay k 8644, a dihydropyridine calcium channel agonist, and U 46619, a thromboxane analogue. Intracoronary bolus administration of endothelin reduced coronary blood flow and increased coronary arterial resistance. Similarly, intracoronary bolus administration of equipotent doses of Bay k 8644 or U 46619 significantly reduced coronary blood flow and increased coronary arterial resistance. The coronary vasoconstrictor effects of endothelin were long-lasting as compared with the transient actions of Bay k 8644 and U 46619. Intracoronary bolus injection of endothelin also reduced left ventricular (LV) dP/dt arterial pressure (MAP), and cardiac output (CO). In contrast, Bay k 8644 increased LVdP/dt but did not alter CO or MAP. Intracoronary bolus injection of U 46619 did not affect MAP, CO, or LVdP/dt. In a separate group of animals, intracoronary infusion of nitrendipine significantly increased coronary blood flow and reduced coronary arterial resistance. Other cardiovascular parameters measured were not significantly altered. In the presence of nitrendipine, the effects of intracoronary administration of endothelin and U 46619 on coronary blood flow, coronary arterial resistance, and LVdP/dt were only partially antagonized. On the other hand, the effects of Bay k 8644 were completely prevented in the presence of nitrendipine. These studies show that at doses which reduce coronary blood flow to the same extent, only endothelin produces myocardial depression in anesthetized dogs. The cardiovascular actions of endothelin were only partially antagonized by nitrendipine, suggesting that mechanisms other than calcium influx through voltage-operated channels are involved.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1992

Bradykinin-induced, N omega-nitro-L-arginine-insensitive endothelium-dependent relaxation of porcine coronary arteries is not mediated by bioassayable relaxing substances.

The effect of the arginine analogue, N omega-nitro-L-arginine (L-NNA) was studied on bradykinin-induced relaxation in porcine coronary arteries. In the presence of indomethacin (3 x 10(-6) M) and captopril (10(-6) M), treatment with L-NNA (10(-4) M) had no effect on the bradykinin-induced (10(-10)-10(-7) M) relaxations in strips contracted with U-46619. In contrast to the findings in organ chamber experiments, bradykinin-induced release of endothelium-derived relaxing factor(s) (EDRFs) was abolished after 45 min of treatment of perfused porcine coronary artery segments with L-NNA (10(-4) M) in a superfusion bioassay system. These results show that, in addition to the release of nitric oxide, endothelium-dependent relaxation of porcine coronary arteries to bradykinin involves an alternative mechanism(s), which accounts for the relaxation in the presence of L-NNA. Since the release of a relaxing mediator could not be detected from L-NNA-treated porcine coronary artery segments under bioassay conditions, it is postulated that either no diffusible factor(s) is involved in the L-NNA-insensitive endothelium-dependent relaxation, or it is mediated by an extremely labile endothelium-derived substance(s).

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗